The overall objective of this research project is to study the mechanism of membrane biogenesis and membrane selectivity. Interest in the study of sphingolipids has been stimulated by recent observations relating these lipids to intercellular recognition, immunological determination and receptors for some toxins and hormones. The specific purpose of this project will be to continue with our studies on the mechanism(s) of synthesis of sphingosine, phytosphingosine and the in vivo pathway(s) for the biosynthesis of sphingomyelins and glucosyl ceramides. Studies with hamster cells will be continued and extended to neuroblastoma cells and chick embryo fibroblasts grown in tissue culture, since a combination of such systems will allow one to determine the generality of any observations. We will complete the chemical synthesis of C18-dihydrosphingosine-containing ceramides labeled with different radioisotopes in both the long chain base and fatty acid moieties. These compounds, as well as radiolabeled C18-phytosphingosine and 3-keto sphingosine and their respective ceramides will be tested by pulse-chase kinetics and specific activity analyses as possible precursors of sphingosine and sphingosine-containing sphingolipids. Attempts will be made to develop a cell free system with rat liver for those reactions that seem to occur in vitro. We will also study whether the in vivo synthesis of glucosyl ceramides occurs via condensation of ceramides with UDP-glucose or via condensation of glucosylsphingosine with a fatty acyl CoA and whether ceramides are in vivo precursors of sphingomyelin in the above mentioned cells. These studies will constitute the groundwork for further investigations on 1. the subcellular localization of enzymes related to the biosynthesis of sphingolipids, 2. attempts to solubilize and purify these enzymes and 3. efforts to modify the sphingolipid composition of cells in culture, enabling one to correlate changes in composition with alterations in proposed functions ascribed to sphingolipids.